Compositions and methods for modulating the immune system

ABSTRACT

The present invention provides methods and compositions for the prophylaxis of blood cell disorders such as neutropenia, thrombocytopenia, lymphocytopenia, and anaemia. The invention provides methods wherein compositions comprising at least one cytokinin compound are administered either therapeutically or prophylactically. The invention further has utility in methods of DNA repair.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of U.S. Pat. No. 8,076,311, filed Dec. 8, 2008,which is the U.S. national phase of PCT/EP2007/054605, filed May 11,2007, which claims the benefit of U.S. Provisional Application No.60/810,961 filed Jun. 5, 2006. The entire disclosure of application Ser.No. 12/227,256 is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods for stimulating the productionof cells of white blood cells. In particular, the present inventionprovides compositions and methods for the enhancement of blood cellproduction. Specifically, the use of the compounds, compositions andmethods of the invention can be used to ameliorate or treat conditionssuch as neutropenia, thrombocytopenia, anaemia and other blood celldeficiencies or other exemplified conditions such as reactive oxidativestress which result from or are caused by gamma x-ray or protonradiation exposure.

BACKGROUND TO THE INVENTION

The suppression or dysregulation of the immune system can be a componentof many pathological diseases or conditions. In particular, immunedysregulation can result in the onset and progression of disease. Whereonset of disease occurs, the dysregulation of the immune system meansthat an individual is compromised against mounting a full immuneresponse against a particular disease or pathogenic condition.

Acute Radiation Syndrome (ARS) results from the exposure of anindividual to a high level of radiation. ARS is a life threateningcondition, primarily due to the depletion in the blood cell count. Highlevels of radiation inhibits production of blood cells by the bonemarrow. Such blood cells include; white blood cells, neutrophils,platelets and factors required for blood clotting. Severe depletion ofthese blood components results in conditions such as neutropenia, wherea low neutrophil count is present and thrombocytopenia where there is alow platelet count.

Although the bone marrow can functionally revert to produce blood cellcomponents at the level produced prior to radiation exposure, thisreversion can take up to 3 months. In the interim, the immune system isseverely compromised due to the depletion in cell numbers, and inparticular due to a depleted neutrophil count.

With the possibility of a terrorist attack which would include, in someform, a radioactive element, there is the need for viable treatments toARS which are cost effective and which are easy to produce andadminister, and which have previous human use for other indications.

Chemotherapy can also cause damage to bone marrow and accordingly resultin a decrease in neutrophil and platelet count. Again, the use ofcompounds which could be administered to reduce the side effects ofchemotherapy on bone marrow cell production would be highly desirable.

The inventor of the present invention has surprisingly identified twogroups of compounds which have been unexpectedly shown to mediate,either on their own, or in combination with other compounds, aprotective effect against blood cell depletion. Preventing blood celldepletion can mediate the remission of neutropenia, thrombocytopenia andanaemia, and can also stimulate an enhancement of B cell and T cellfunction, following such function being compromised due to exposure of asubject to radiation.

The compounds identified by the inventor are non steroidal compounds,which have been unexpectedly shown to have utility in the treatmentand/or amelioration of one or more condition which can be attributed toa deficiency or dysregulation in blood cell number, and in particular ofthe function of the immune system. This dysregulation is particularlycharacterised by a reduction in a reduction in a number of cells, suchas lymphocytes which have a role or function in mediating immunity. Assuch, the compounds of the present invention have utility in methods forthe treatment of immuno-suppressive conditions mediated by exposure toradiation, and in particular to blood cell deficiencies such asneutropenia and thrombocytopenia.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided amethod for the treatment and/or prophylaxis of blood cell depletion, themethod comprising:

-   -   providing a therapeutically effective or prophylactically        effective amount of oltipraz or a derivative, analogue,        metabolite, prodrug or pharmaceutically acceptable salt thereof,        and    -   administering the same to a subject in need of such treatment.

In certain embodiments, the blood cell depletion results in immunedysregulation. In certain further embodiments, the blood cell depletionresults in a condition selected from, but not limited to; neutropenia,thrombocytopenia, lymphocytopenia, and anaemia.

As herein defined, neutrotopenia is a condition characterised by adecrease in the number of neutrophils. Lymphocytopenia is a conditioncharacterised by a decrease in the number of lymphocytes.Thrombocytopenia is a condition characterised by a decrease in thenumber of platelets.

In certain embodiments, the neutropenia is selected from the groupconsisting of, but not limited to: postinfectipous neutropenia,autoimmune neutropenia, chronic idiopathic neutropenia or a neutropeniaresulting from, or potentially resulting from, chemotherapy for use inthe treatment of a cancerous condition, chemotherapy for the treatmentof an autoimmune disease, an antiviral therapy, direct radiationexposure, secondary radiation exposure through environmentalcontamination, tissue or solid organ allograft or xenograft rejection orimmune suppression therapy in tissue or solid organ transplantation oraging or immunesenescence.

In certain further embodiments, the condition is reactive oxidativestress, for example, reactive oxidative stress which may result from, orwhich may be caused by exposure of a subject to gamma, x-ray or protonradiation exposure.

The structure of oltipraz is shown in Formula I:

In certain further embodiments, the metabolite of oltipraz (also knownas the dithiolethione oltipraz (OPZ)) is metabolite 3 (also known asM3). Oltipraz undergoes metabolism by molecular rearrangement to yieldM3, a pyrrolopyrazine derivative.

In certain further embodiments, oltipraz or a derivative, analogue,metabolite or prodrug thereof is formulated along with carboxymethylcellulose (CMC) to form a combined medicament. Carboxymethyl celluloseis a cellulose derivative with carboxymethyl groups bound to some of thehydroxyl groups of the glucopyranose monomers that make up the cellulosebackbone.

The inventor has surprisingly identified that formulating oltipraz alongwith carboxymethyl cellulose results in a marked reduction in toxicity.Specifically, and without wishing to be bound by theory, the inventorhas identified that when formulated this way, oltipraz is safe foradministration to a subject in an amount of 2000 mg/kg. The inventor hasidentified that oltipraz, when administered individually, is expected tocause toxicity in the liver at levels of 50 to 100 mg/kg. The inventorhas identified that when formulated with carboxymethyl cellulose,oltipraz is not absorbed into the bloodstream, but rather lines thedigestive tract, this serving to protect against damage such asgastrointestinal damage.

In a further aspect of the present invention there is provided apharmaceutical composition for use in the treatment of blood celldepletion, the composition comprising oltipraz or a derivative,analogue, metabolite, prodrug or pharmaceutically acceptable saltthereof, along with a pharmaceutically acceptable diluent and/orcarrier.

Typically the pharmaceutically acceptable diluent or carrier will beselected depending upon the intended route of administration of thepharmaceutical composition.

In certain embodiments, the composition comprises the oltiprazderivative M3 (metabolite 3).

In certain embodiments, the pharmaceutical composition further comprisescarboxymethyl cellulose.

A further aspect of the invention provides for the use of oltipraz or aderivative, analogue, metabolite, prodrug or pharmaceutically acceptablesalt thereof in the preparation of a medicament for the treatment ofblood cell disorder.

In certain embodiments, the blood cell disorder is selected from thegroup consisting of, but not limited to; neutropenia, thrombocytopenia,lymphocytopenia, and anaemia.

In certain embodiments, the derivative of oltipraz is M3 (metabolite 3).In certain further embodiments, the composition further comprisescarboxymethyl, cellulose.

A still further aspect of the present invention provides a kitcomprising oltipraz or a derivative, analogue, metabolite, prodrug orpharmaceutically acceptable salt thereof for use in preventing bloodcell depletion.

Without wishing to be bound by theory, the inventor predicts, that themode of action is derived from oltipraz or a derivative or analoguethereof chelating with, or forming a complex with, one or more divalentor trivalent radioactive metal ions, whereby the divalent or trivalentradioactive ions in the subject's cells or tissues are redistributed orsequestered such that the ions are limited in their capacity toparticipate in unwanted tissue destruction.

In certain embodiments, the divalent or trivalent metal ions areselected from the group comprising, but not limited to: Fe, Cu, Ni, Ca,Mg, Mn, Cd, Pb, Al, Hg, Co, I, Se, Cs, U, Pa, Th, Ra, Ce, and Zn.

According to a further aspect of the present invention there is provideda method for the treatment and/or prophylaxis of blood cell depletion,the method comprising:

-   -   providing a therapeutically effective or prophylactically        effective amount

of at least one cytokinin compound, and

-   -   administering the same to a subject in need of such treatment.

In certain embodiments, the blood cell depletion results in immunedysregulation. In certain further embodiments, the blood cell depletionresults in a condition selected from, but not limited to; neutropenia,thrombocytopenia, lymphocytopenia, and anaemia.

As herein defined, neutrotopenia is a condition characterised by adecrease in the number of neutrophils. Lymphocytopenia is a conditioncharacterised by a decrease in the number of lymphocytes.Thrombocytopenia is a condition characterised by a decrease in thenumber of platelets.

In certain embodiments, the neutropenia is selected from the groupconsisting of, but not limited to; postinfectious neutropenia,autoimmune neutropenia, chronic idiopathic neutropenia or a neutropeniaresulting from, or potentially resulting from, chemotherapy for use inthe treatment of a cancerous condition, chemotherapy for the treatmentof an autoimmune disease, an antiviral therapy, direct radiationexposure, secondary radiation exposure through environmentalcontamination, tissue or solid organ allograft or xenograft rejection orimmune suppression therapy in tissue or solid organ transplantation oraging or immunesenescence.

In certain further embodiments, the condition is reactive oxidativestress, for example, reactive oxidative stress which may result from, orwhich may be caused by exposure of a subject to gamma, x-ray or protonradiation exposure.

In certain embodiments, the cytokinin compound is N⁶ isopenteriyladenosine or an analogue, derivative, metabolite, prodrug orpharmaceutically acceptable salt thereof.

In a further embodiment, the method further comprises the step ofadministering to the subject a therapeutically effective amount ofoltipraz or a derivative, analogue, metabolite, prodrug orpharmaceutically acceptable salt thereof.

Examples of N⁶ isopentenyl adenosine or an analogue, derivative,metabolite, prodrug or pharmaceutically acceptable salt thereof areshown below, said compounds being described hereinafter.

In certain embodiments, N⁶ isopentenyl adenosine compounds according toFormula 2 are provided:

wherein:R1═H, R2═CH3, R3═CH3, and R4═H, or R1═H or CH3S and R4 is as follows:

and R5═CH3, C1, OH or a monophosphate group, R6═CH3, CH2OH or Cl, andR7═H or Br, or R1═H and R4 is as follows:

and X1 and X2 are independently selected from H, methyl, ethyl,hydroxyl, a halogen and carboxyl or R4 is as follows:

and wherein R8 is as follows:

and R2═OH and R3═OH, monophosphate, diphosphate or triphosphate group,or R2 and R3 are linked to form a 3′,5′-cyclic monophosphate derivative,or a physiologically acceptable salt of any such compound.

Formula II is used herein to refer to all of such compounds and salts.

This aspect of the invention further extends to further N⁶ isopentenyladenosine compounds as described below. These compounds are listedhereinafter as compounds Ia through to Iu, wherein said compounds definethe constituents of R1, R2, R3 and R4 of Formula II. The compounds areas follows:

Compound Ia wherein: R1═H, R2═OH, R3═OH and R4 Is as follows:

This compound being known as N6-(Δ2-isopentenyl) adenosine.

Compound Ib wherein: R1═H, R2═OH, R3=monophosphate and R4 is as follows:

This compound being known as N6-(Δ2-isopentenyl)adenosine-5′-monosphosphate.

Compound Ic wherein: R1═H, R2 and R3 are linked to form a 3′,5′-cyclicmonophosphate derivative, and R4 is as follows:

This compound being known as N6-(Δ2-isopentenyl) adenosine-5′-cyclicmonosphosphate.

Compound Id wherein: R1═H, R2═OH, R3═OH and R4═CH2C66, the compoundbeing known as N6-benzyladenosine.

Compound Ie wherein: R1═H, R2═OH, R3═monophosphate, and R4═CH2C6H6. Thecompound being known as N6-benzyladenosine-5′-monophosphate.

Compound If wherein: R1═H, R2 and R3 are linked to form a 3′,5′-cyclicmonophosphatederivative and R4═CH2C6H6. Wherein the compound is known asN6-benzyladenosine-3′,5′byclic monophosphate.

Compound Ig, wherein: R1═H, R2═OH, R3═OH, and R4 is as follows:

Wherein the compound is known as Furfuryladenosine.

Compound Ih which is N6-furfuryladenosine-5′monophosphate. Compound Iiwhich is N6-furfuryladenosine-3′,5′-cyclic monophosphate. Compound Ijwhich is N-(purin-6-ylcarbamoyl)-o-chloroaniline ribonudeoside. CompoundIk which is N-(purin-6-ylcarbamoyl)-o-chloroanilineribonucleoside-5′monophosphate. Compound II which isN6-adamantyladeriosine. Compound Im which isN6-adamantyladenosine-5′-monophosphate. Compound In which isN-(purin-6-ylcarbamoyl)-n-octylamine ribonudeoside. Compound lo which isN-(purin-6-ylcarbamoyl)n-octylamine ribonucleoside-5′-monophosphate.Compound Ip which is N-(purin-6-ylcarbambyl)-n-octylamineribonucleoside-3′,5′-cyclic monophosphate, Compound Iq which isN6-(Δ2-isopentyl)-2-methylioadenosine. Compound Ir which isN6-(4-hydroxy-3-methyl-trans-2-butenyl)-adenosine. Compound Is which isN6-(3-chloro-trans-butenyl) adenosine. Compound It which isN6-(3-chloro-cis-2-butenyl) adenosine. Compound Iu wherein: R1═H,R2═CH3, R3═CH3 and R4═H.

The present invention further extends to one or more metabolites of thecompounds of Formula II. For example, preferred metabolites include:n6(Δ2-isopentenyl) adenine, 6-N-(3-methyl-3-hydroxybutylamino) purine,adenine, hypoxathine, uric acid and methylated xanthines.

In certain further embodiments, N⁶ isopentenyl adenosine or aderivative, analogue, metabolite or prodrug thereof is formulated alongwith carboxymethyl cellulose (CMC) to form a combined medicament.Carboxymethyl cellulose is a cellulose derivative with carboxymethylgroups bound to some of the hydroxy groups of the glucdpyranose monomersthat make up the cellulose backbone.

The inventor has surprisingly identified that formulating N⁶ isopentenyladenosine along with carboxymethyl cellulose results in a markedreduction in toxicity. Specifically, and without wishing to be bound bytheory, the inventor has identified that when formulated this way, N⁶isopentenyl adenosine is safe for administration to asubject in anamount of 2000 mg/kg. The inventor has identified that N⁶ isopentenyladenosine, when administered individually, is expected to cause toxicityin the liver at levels of 50 to 100 mg/kg. The inventor has identifiedthat when formulated with carboxymethyl cellulose, N⁶ isopentenyladenosine is not absorbed into the bloodstream, but rather lines thedigestive tract, this serving to protect against damage such asgastrointestinal damage.

In a further aspect of the present invention there is provided apharmaceutical composition for use in the treatment of blood celldepletion, the composition comprising N⁶ isopentenyl adenosine or aderivative, analogue, metabolite, prodrug or pharmaceutically acceptablesalt thereof, along with a pharmaceutically acceptable diluent and/orcarrier.

Typically the pharmaceutically acceptable diluent or carrier will beselected depending upon the intended route of administration of thepharmaceutical composition.

In certain embodiments, the pharmaceutical composition further comprisescarboxymethyl cellulose.

A further aspect of the invention provides for the use of N⁶ isopentenyladenosine or a derivative, analogue, metabolite, prodrug orpharmaceutically acceptable salt thereof in the preparation of amedicament for the treatment of blood cell disorder.

In certain embodiments the N⁶ isopentenyl adenosine is a compoundaccording to Formula II.

In certain embodiments, the blood cell disorder is selected from thegroup consisting of, but not limited to; neutropenia, thrombocytopenia,lymphocytopenia, and anaemia.

A still further aspect of the present invention provides a kitcomprising N⁶ isopentenyl adenosine or a derivative, analogue,metabolite, prodrug or pharmaceutically acceptable salt thereof for usein preventing blood cell depletion.

In certain embodiments the N⁶ isopentenyl adenosine is a compoundaccording to Formula II.

In certain further embodiments, the cytokinin compound is N⁶ benzyladenosine or an analogue, derivative, metabolite, prodrug orpharmaceutically acceptable salt thereof.

Examples of N⁶ benzyl adenosine or an analogue, derivative, metabolite,prodrug or pharmaceutically acceptable salt thereof are shown below,said compounds being described hereinafter.

In certain further embodiments, the N⁶ benzyl adenosine isN6-Benzyl-adenosine-5′monophosphate, which is shown below as a compoundhaving Formula III:

In certain further embodiments, the N⁶ benzyl adenosine is(N6-Benzyl)Adenyl-p-(N6-Benzyl)Adenyl-p-(N6Benzyl) Adenosine, which isshown below as a compound having Formula IV:

In certain further aspects, the present invention further extends to thefollowing compounds:

ADT, having the general structure:

ADO, having the general structure:

1,2-Dithiole 3-thione having the structure:

Lipoamide (1,2-dithiolane), having the structure:

1,3-dithiole 2-thione having the structure:

[1,2]Dithiolo[4,3-c]-1,2-dithiole-3,6-dithione having the structure:

Malotilate having the general structure:

1,2-Dithiolane, class 1, having the general structure:

1,2-Dithiole, class 2, having the general structure:

1,3-Dithiole, class 3, having the general structure:

1,3-Dithioloane, class 4, having the general structure:

wherein Z═S, O, NR, R2, CR2 and Z can have the designations optionallyand independently for allthe classes. R in this case includes, H, alkyl(C1-C5), alkoxy (C1-C5), alkoxycarbononyl (C1-C5). R2 can form spirorings about the ring carbon atom.

For the thiolane classes, the ring carbon atoms can be doublysubstituted. R1-R4 are the main ring substituents for all classes and inorder to cover a wide variety of substituents, should include optionallyand independently H, alkyl, aryl, heterocyclic, halogen, alkoxycarbonyl(C1-C5) or carboxyl.

R1, R2 or R3, R4 can form a spiro ring around the carbon atom to whichthey are attached or they can form fused or bridged rings to adjacentcarbons atoms. The following definitions cover the majority ofcompounds.

Alkyl is defined herein as C1-CT0 linear or branched chain, saturated orunsaturated which can optionally be singly or multiply substituted byhalogen, alkyl (C1-C5), hydroxyl, alkoxy (C1-C5), alkoxycarbonyl,(C1-C5), carboxyl, amido, alkyl amido (C1-C5), amino, mono and dialkylamino (C1-C5), alkyl carbamoyl (C1-C5) thiol, alkythio (C1-C5) orbenzenoid aryl.

Aryl is herein defined as any optionally singly or multiply substitutedbenzenoid group (C6-C14).

Heterocyclic radical means any 4, 5 or 6 membered, optionallyhetercyclic ring, saturated or unsaturated, containing 1-3 ring atoms ofwhich N, O or S, the remaining atoms being carbon.

Substituents on the aryl or heterocyclic radical include:

halogen, alkyl (C₁-C₅), hydroxyl, alkoxy (C₁-C₅), alkoxycarbonyl,(C₁-C₅), carboxyl, amido, alkylamido (C₁-C₅), amino, mono and dialkylamino (C₁-C₅), alkyl carbamoyl (C₁₋C₅), thiol, alkyl thio (C₁₋C₅) orbenzenoid aryl, cyano, nitro, halo alkyls, alklsulfonyl (C₁₋C₅),sulfonate. Two of such substituents can be part of a fused ring, whichcan be either saturated, or unsaturated, heterocyclic or carbo cyclic.

in which

X is chosen from

═S

═O

═N—OH

═N—R₅

R₅, being a C₁-C₆ alkyl or an aryl group,

═N—NH—CO—NH₂ and

═N—NH—CS—NH₂ and

Z and Z′ being electron-attracting groups such as ester or cyano groups.

A is chosen from a >C═N—OH group, a group of formula >C═N—OR₃ (where R₃is chosen from hydroxyl, amino, chloro and C₁-C₄, alkoxy groups, an aryl(C₁-C₆ alkyl) group, a (C₁-C₆ alkyl) Carbonyl group and an aryl (C₁-C₆alkyl) earbonyl group).

A may also be chosen from a >C═O group, a >C═N—R₄ group, R₄ being aC₁-C₆ alkyl group or an aryl group, and a CHOH group.

R₁ and R₂ are chosen, independently of one another, from hydrogen, ahalogen, a nitro group, a nitroso group, a thiocyano group, a C₁₋C₆alkyl group, a C₂-C₆ alkenyl group, an aryl group, aryl (C₁-C₆ alkyl)group, an aryl (C₂₋C₆ alkenyl) group, a carboxyl group, a (C₁-C₆ alkyl)carbonyl group, an arylcarbonyl group, a (C₁-C₆ alkoxy) carbonyl, group,a (C₁-C₆ alkoxy) carbonyl (C₁-C₆ alkyl) group, a C₁-C₆ alkoxy group, atrifluoromethyl group, an amino group, a di (C₁-C₆ alkyl) amino (C₁-C₆alkyl), an acylamino group of formula —NHCOC_(n)H_(2n+1) with n from 0to 6, a group —NH—CSC_(n)H_(2n+1) with n from 0 to 6, a terpenyl group,a cyano group, a C₂-C₆ alkynyl group, a C₂-C₆ alkynyl group substitutedwith a C₁-C₆ alkyl or an aryl group, a hydroxy (C₁-C₆ alkyl) group, a(C₁-C₆ acyl) oxy (C₁-C₆ alkyl) group, a (C₁-C₆ alkyl) thio group and anarylthio group;

or alternatively R₁ and R₂ together form a mono- or polycyclic C₂-C₂₀alkylene group optionally comprising one or more hetero atoms, with theexception of the 2,2dimethyltrimethylene group, or a C₃-C₁₂cycloalkylene group.

R is chosen from C₁-C₆, alkyl group, and their pharmaceuticallyacceptable salts.

In the foregoing definition, aryl group or aryl fraction of an arylalkylgroup denotes an aromatic carbon-based group such as a phenyl ornaphthyl group or an aromatic heterocyclic group such as a thienyl offuryl group, it being possible for these groups to bear one or moresubstituents chosen from a halogen atom, a C₁-C₄ alkyl group, a C₁-C₄alkoxy group, a trifluoromethyl group, a nitro group and a hydroxylgroup.

Oximes of 1,2-dithiole-3-thione derivatives such as a shown as follows:

Additionally Aldehydes or Ketones of previously identified compounds areincorporated such as shown as follows:

one or more of the following compounds according to an embodimentwherein A (Formula V, VI) is a group C═N═OR′₃ where R₃ is an optionallysubstituted C₁-C₆ alkyl group, in particular substituted with one ormore groups chosen from hydroxyl, amino, chloro, bromo, fluro, iodo andC₁-C₄ alkoxy groups, or an aryl (C₁-C₆ alkyl) group, that is to saycompounds of formula

in which R₃ has the meaning given above

one or more of the following compounds as described in an embodiment(Formula V, VI) in which A is a group C═N—O—CO—R″₃, R″₃ being chosenfrom a hydrogen atom, an optionally substituted C₁-C₆ alkyl group, anaryl group and an aryl (C₁-C₆ alkyl) group, that is to say compounds ofthe following formula:

in which R″₃ being chosen from a hydrogen atom, an optionallysubstituted C₁-C₆ alkyl group, an aryl group.

Another group of compounds is formed by the compounds of embodiment(Formula V, VI) in which A is a CH—OH group, that is to say thecompounds of the following formula:

Another group of compounds is formed by the compounds of embodiment(Formula V, VI) in which A is a group C═N—R, R being a C₁-C₆ alkyl or anaryl group, that is to say compounds of the following formula:

Another group of compounds include compounds of embodiment (Formula V,VI) in which A is a C═O group and X is an oxygen atom, that is to saycompounds of the following formula:

in which

R₁ is chosen from hydrogen, a halogen, a nitro group, a nitroso group, athiocyano group, a C₁-C₆ alkyl group, a C₂-C₆ alkenyl group, an arylgroup, an aryl (C₁-C₆ alkyl) group, an aryl (C₂-C₆ alkenyl) group, acarboxyl group, a (C₁-C₆ alkyl) carbonyl group, an arylcarbonyl group, a(C₁-C₆ alkoxy) carbonyl group, a (C₁-C₆ alkoxy) carbonyl (C₁-C₆ alkyl)group a (C₁-C₆ alkoxy group, a trifluoromethyl group, an amino group, adi (C₁-C₆ alkyl) amino (C₁-C₆ alkyl) group, an acylamino group offormula —NHCOC_(n)H_(2n+1) with n from 0 to 6, a group—NH—CSC_(n)H_(2n+1) with n from 0 to 6, a terpenyl group, a cyano group,a C₁-C₆ alkynyl group, a C₂-C₆ alkynyl group substituted with a C₁-C₆alkyl or an aryl group, a hydroxy (C₁-C₆ alkyl) group, a (C₁-C₆acyl)-oxy(C₁-C₆ alkyl) group, a C₁-C₆ alkyl)thio group and an arylthiogroup.

R₂ is chosen from a nitro group, a nitroso group, a thiocyano group, aC₁-C₆ alkyl group, a C₂-C₆ alkenyl group, an aryl group; an aryl (C₁-C₆alkyl) group, an aryl (C₁-C₆ alkenyl) group, a carboxyl group, a (C₁-C₆alkyl)carbonyl group, an arylcarbonyl group, a (C₁-C₆ alkoxy)carbonylgroup, a (C₁-C₆ alkyl) group, a trifluoromethyl group, a di(C₁-C₆alkyl)amino(C₁-C₆ alkyl) group, an acylamino group of formula—NHCOC_(n)H_(2n+1) with n from 0 to 6, a group —NH—CSC_(n)H_(2n+1) withn from 0 to 6, a terpenyl group, a cyaho group, a C₂ -C₆ alkynl group, aC₂-C₆ alkynyl group substituted with a C₁-C₆ alkyl or an aryl group, ahydroxy (C₁-C₆ alkyl) group, a C₁-C₆ acyl-oxy(C₁-C₆ alkyl) group, a(C₁-C₆ alkyl)thio group and an arylthio group;

or alternatively R₁ and R₂ together form a mono- or polycyclic C₂-C₂₀alkylene group optionally comprising one or more hetero atoms,

one or more of the compounds of the following formula:

R₁ and R₂ are chosen, independently of one another, from hydrogen, ahalogen, a nitro group, a nitroso group, a thiocyano group, a C₁-C₆alkyl group, a C₂-C₆ alkenyl group, an aryl group, aryl(C₁-C₆ alkyl)group, an aryl (C₂-C₆ alkenyl) group, a carboxyl group, a (C₁-C₆alkyl)carbonyl group, an arylcarbonyl group, a (C₁-C₆ alkoxy)carbonylgroup, a (C₁-C₆ alkoxy)carbonyl (C₁-C₆ alkyl) group, a C₁-C₆ alkoxygroup, a trifluoromethyl group, a di(C₁-C₆ alkyl)amino(C₁-C₆ alkyl)group, an acylamino group of formula —NHCOC_(n)H_(2n+1) with n from 0 to6, a group —NH—CSC_(n)H_(2n+1) with n from 0 to 6, a terpenyl group, acyano group, a C₂-C₆ alkynyl group, a C₂-C₆ alkynyl group substitutedwith a C₁-C₆ alkyl or an aryl group, a hydroxy(C₁-C₆ alkyl) group, a(C₁-C₆ acyl) oxy (C₁-C₆ alkyl) group, a (C₁-C₆ alkyl) thio group and anarylthio group;

or alternatively R₁ and R₂ together form a mono- or polycyclic C₂-C₂₀alkylene group optionally comprising one or more hetero atoms.

R is chosen from a C₁-C₆ alkyl group, and their pharmaceuticallyacceptable salts.

In the foregoing definition, aryl group or aryl fraction of an arylalkylgroup denotes an aromatic carbon-based group such as a phenyl ornaphthyl group or an aromatic heterocyclic group such as a thienyl offuryl group, it being possible for these groups to bear one or moresubstituents chosen from halogen atom, a C₁-C₄ alkyl group, a C₁-C₄alkoxy group, a trifluoromethyl group, a nitro group and a hydroxylgroup,

one more of the following isobenzothiazolone derivative having thestructure:

In this structure at least one of R¹ and R² is preferably nitro,arylazo, substituted arylazo, benzylideneamino or substitutedbenzylideneamino. When only one of R¹ and R² is so substituted, one ofR¹ and R² may be hydrogen. The R³ substituent is selected from alkylgroups in less than about 7 carbon atoms, amino, hydroxyl, alkoxyl, andaryl groups (and functidnalized forms thereon).

Preferred species of the isobenzothiazole derivative of the presentinvention comprise R¹ as nitro or arylazo and R² as hydrogen, forexample. Examples include compounds where R² is hydrogen and R¹ isphenylazo; substituted arylazo such as 4-hydroxyphenylazo; 4nitro-2-methylphenylazo; 2-hydroxy-1-napthylazo;2-hyroxy-5-methylphenylazo; 2-hydroxy-4-methyl-5-nitrophenylazo;4-hydroxy-1-napthylazo; 4-hydroxy-3-methyl-1-napthylazo;4-hydroxy-5-aza-1-napthylazo; 2amino-1-napthylazo;1-hydroxy-2-napthylazo;3-N,Ndimethylaminopropylcarboxyamido-1-hydroxy-4-naphthylazo;1-hydroxy-4-methoxy-2-naphthylazo, 2-hydroxy-3-carboxy-1-naphthylazo;1-hydroxy-3,6-disulfonato-2-naphthylazo; 2, 3-dihydroxy-1-naphthylazo;or 2-hydroxy-3,5-dimenthyl-1-phenylazo. In one particular embodiment R¹is the substituted benzylideneamino, 2,4-dinitrobenzylideneamino and R²is hydrogen. Additionally R¹ is hydrogen and R² is2-hydroxy-1-naphthylazo or 4-hydroxy-Iphenylazo.

In one aspect, R³ substituents with sufficient polarity to conferaqeuous solubility upon the compound. For example, R³ may be —(CH₂)nR⁴R⁵where n is from 2 to 6 and R⁴ and R⁵ are simple alkyls or hydrogens.Other possible water solubilizing side chains include 3-carboxyprppyl,sulfonatoethyl and polyethyl ethers of the type —CH₂(CH₂OCH₂)CH₃ where nis less than 10. Preferred compounds include R³ side chains containingaminoalkyl, carbdxyalkyl, omega amino polyethyl ethers and N-haloacetylderivatives. In a broader sense, for various utilities R³ may be alkyl,aryl, heteroaryl, alkoxy, hydroxyl or amino groups. When includingsubstitutions for solubility, or reactivity purposes, R³ may beaminoalkyl, carboxyalkyl, hydroxyalkyl or haloalkyl. The aryl orheteroarl R³ moieties may be substituted, for example as aminoaryl,carboxyaryl or hydroxyaryl,

one or more of the following Isobenzothiazolone derivative having thestructure:

wherein at least one of R¹ and R² is nitro, arylazo, substitutedarylazo, benzylideneamino or substituted benzyfideneamino and one of R¹and R² may be hydrogen and R³ is a aminoallayl, aminoaryl andaminoheteroaryl, carboxyalkyl, carboxyafyl or carboxyheteroarylcovalently linked to a polymer comprising amino or hydroxy groups. Thespacer arm R³ can comprise oligmers or polyethylene-glycol and itsderivatives. In one aspect, R³ may be17-chloracetamidd-3,6,9,12,15-pentaoxyheptadecyl where hexaethyleneglycol has been chloroacetamidated. When the polymer groups, Y¹ and R³comprises carboxyl groups, the covalent linkage is preferably through anester bond. When the polymer comprises amino groups, the analog covalentlinkage is through an amide bond. The amine bearing polymer, whencoupled to R³, may be-a polymer such as chitosan, polyalkylamine,aminodextran, polyethyleneimine, polylysine or amitryrene.

The R³ substituents of the present invention may also comprise an alkyllinked to an amine bearing polymer by amine displacement of a halogenfrom an alpha-haloalkyl or alpha-haloalkylcarbox amido R³ precursor. Inthe case of aminoalkyl or aminoaryl groups the R³ substituent may alsobe covalently linked to a polymer such as polyepichlorohydrin,chloromethylpolystyrene, polyvinylalochol or polyvinylpyridine. The R³substituent of the present invention may generally be an aminoalkyl,hydroxyalkyl, aminoaryl or hydroxyaryl group linked to a polymercomprising carboxyl groups through amide or ester linkages.

When polymers are involved in the R³ structure, the polymer may be onesuch as polyacrylic acid, polymethacrylic acid, polyilaconic acid,oxidized polyethylene oxide, poly(methylmethacrylate/methacrylic acid),carboxyinethyl cellulose, carboxymethyl agarose or carboxymethyldextran. When such a carboxyl polymer is involved, the R³ may beaminoalkyl (such as 8 aminohexyl, for example), hydroxyalkyl, aminoarylor hydroxyaryl linked to the polymer through amide or ester linkages. Insuch cases, an R³ precursor function may bear an amine or hydroxyl groupto be covalently linked to a polymer by reaction with an acidanhydride-bearing polymer or by coupling with a carboxylate bearingpolymer through carbodimideinduced bond formation.

The R³ substituent or precursor thereto in the compound of the presentinvention may also be a haloalkyl or carboxylialoalkyl moiety such aschloracetamido. Such a substituent may be readily coupled to an aminebearing polymer by amine displacement of the halogen.

“Aryl,” as used herein, is intended to include organic residues derivedfrom aromatic hydrocarbon or aromatic heterocyclic ring systems.Accordingly aryl groups include the unsubstituted ring residues such asphenyl and naphthyl and substituted forms thereof. Heterocyclic orheteroaryl residues may be those comprising one or more heteroatoms(e.g., nitrogen, oxygen, sulphur) in the ring system such as pyridyl,oxazolyl, quinoly), thiazolyl and substituted forms thereof. “Alkyl” asused herein, is intended to include aliphatic and cyclic organicresidues having a carbon at a point of attachment. Accordingly, alkylgroups include unsubstituted hydrocarbon residues of the formulaC_(n)H_(2n+1) and substituted and cyclic forms thereof. Suchhydrocarbons are usually of the lower alkyl class which have six carbonsor less. It is understood that larger alkyl groups may be used. Alkylincludes substituted residues which are intended to include thehydrocarbon residues bearing one or more, same or different, functionalgroups as described below.

The alkyl and aryl group previously described may be substituted withfunctional groups. Such functional groups include essentially allchemical groups which can be introduced synthetically and result instable compounds. Examples of these functional groups are hydroxyl,halogen (fluoro, chloro, bromo), amino (including alkylamino anddialkylamino), cyano, nitro, carboxy (including carbalkoxy), carbamoyl(including N and N,N alkyl), sulfb, alkoxy, alkyl, aryl, and arylazo,

one or more of the following compounds

wherein R₁ and R₂ are independently (═O) or —OR, where R is H or (C₁-C₄)alkyl; and R₃ is H or (C₁-C₄) alkyl. Preferably, R₃ is H. Preferably R¹and R₂ are (═O) or OH,

one or more of the following compounds

wherein X is H or both X's represent a direct bond between the twosulphur atoms; R₁ is (═O) or —OH; and R₂ is H, Na, K or (C₁-C₄) alkyl.

In particular the compound may be 3-keto lipoic acid, 3-hydroxy lipoicacid, 3-keto dihydrolipoic acid or 3-hydroxy dihydrolipoic acid,

one or more the following

1,2-dithiol-3 thione derivative of a formula shown as follows:

wherein R denotes hydrogen, halogen, lower alkoxy group, lower alkylgroup, amino group, lower alkylsubstituted amino group or loweralkoxycarbonyl group.

In the above-described formula, the term “lower” means methyl, ethyl,propyl and butyl, as well as its structural isomers such as isopropyl,isobutyl and tertiarybutyl.

Among the compounds of the above-described formula, preferred compoundsinclude

5-(4-phenyl-1,3-butadienyl)-1,2-dithiol-3-thione,

5-4(4-chlbrophenyl)-1,3-butadienyl-1,2-dithiol-3-thione,

5-{4(4-methoxyphenyl)-1,3-butadienyl}-1,2-dithiol-3-thione,

5-{4-(p-toluyl)-1,3-butadienyl}-1,2-dithiol-3-thione,

5-{4-(o-chlorophenyl)-1,3-butadienyl}-1,2-dithiol-3-thione, and

5-{4-(m-(methylpheny)-1,3-butadienyl}-1,2-ffithiol-3-thione.

The following compounds are also included:

one or more of the following;

1,2-dithiole of the following formula:

Wherein Het represents pyrimidin-2-yl, pyrimidin-4-yl, orpyrimidin-5-yl, or a said pyrimidin-2-yl, pyrimidin4-yl orpyrimidin-5-yl substituted by halogen, alkyl of 1 through 4 carbonatoms, alkoxy of 1 through 4 carbon atoms, mecapto, alkylthio of 1through 4 carbon atoms, or dialkylamino having 1 through 4 carbon atomsin each alkyl, and R represents halogen, alkyl of 1 through 4 carbonatoms, alkyl of 1 through 4 carbon atoms substituted by alkoxycarbonylhaving 1 through 4 carbon atoms in the alkoxy, carboxy, alkoxycarbonylhaving 1 through 4 carbon atoms in the alkoxy, carbamoyl,N-alkylcarbamoyl having 1 through 4 carbon atoms in the alkyl, orR—CH(OH)— in which R represents hydrogen or alkyl of 1 through 3 carbonatoms.

Without wishing to be bound by theory, the inventor predicts, that themode of action is derived from N⁶ isopentenyl adenosine or a derivativeor analogue thereof chelating with, or forming a complex with, one ormore divalent or trivalent radioactive metal ions, whereby the divalentor trivalent radioactive ions in the subject's cells or tissues areredistributed or sequestered such that the ions are limited in theircapacity to participate in unwanted tissue destruction.

In certain embodiments, the divalent or trivalent metal ions areselected from the group comprising, but not limited to: Fe, Cu, Ni, Ca,Mg, Mn.Cd, Pb, Al, Hg, Co, I, Se, Cs, U, Pa, Th, Ra, Ce, and Zri.

In certain embodiments, the eytokinin is a prodrug of a cytokininprovided by the invention, wherein said prodrug is converted into abiologically active or effective compound by metabolism or hydrolysis.In certain further embodiments, said cytokinin compound is furthermetabolised to form a metabolite, said metabolite mediating the effectof the present invention.

As herein defined, the term “cytokinin” means a compound which is aplant growth substance (plant hormone) which is involved in cell growthand differentiation as well as in other processes. In particular theterm encompasses the class of cytokinins termed “adenine cytokines”which include kinetin, zeatin and benzyl adenine. The term furtherincludes “phenylurea cytokinins” such as N,N′-diphenylurea, whichalthough having a differing chemical composition, has a similarbiological activity to “adenine cytokinins.

Suitable cytokinin compounds for use in the foregoing aspects.of thepresent invention are provided here as Formula 1 compounds. Examples ofsuitable Formula 1 compounds are detailed herein in annex 1.

In the embodiments of the invention, wherein the method of this aspectof the invention is performed prophylactically, typically said method isperformed prior to the exposure of the subject to an insult, such as achemical insult, which may result in blood cell number depletion. Incertain further embodiments, the insult is a biological insult, aradiation insult or a combination thereof, wherein said insult mayinduce or cause the progression of blood cell depletion and the ensuingdevelopment of a condition such as anaemia, neutropenia orthrombocytopenia.

Where a patient is treated in accordance with the method of this aspectof the present invention, it is preferred that the treatment reduces (1)the severity of pain during vascular or microvascular occlusions, (2)the severity of vascular or microvascular occlusions or (3) thefrequency of vascular or microvascular occlusions.

In one embodiment the composition which is administered during themethod of the foregoing aspect of the invention comprises at least onecytokinin compound along with at least one pharmaceutically acceptablecarrier or diluent.

Further provided is the use of the combined medicament or apharmaceutical composition comprising the same in the performance of themethods of the present invention for the prophylaxis and/or treatment ofa blood cell disorder.

Accordingly, a further aspect of the present invention provides a methodfor the prevention and/or treatment and/or amelioration of a blood celldeficiency disorder comprising:

-   -   providing a therapeutically effective amount of oltipraz, and    -   administering the same to a subject in need of such treatment.

In certain embodiment, the blood cell disorder is selected from thegroup comprising, but not limited to, neutropenia, thrombocytopenia oranaemia.

The method of this aspect of the invention has further utility in thetreatment of symptoms of neutropenia, thrombocytopenia or anaemia, andaccordingly the cytokinin compounds of the invention may be administeredto individuals in order to treat such conditions.

In certain embodiments, the neutropenia is postinfectious neutropenia,autoimmune neutropenia, chronic idiopathic neutropenia or a neutropeniaresulting from or potentially resulting from a cancer chemotherapy,chemotherapy for an autoimmune disease, an antiviral therapy, directradiation exposure, secondary radiation exposure through environmentalcontamination, tissue or solid organ allograft or xenograft rejection orimmune suppression therapy in tissue or solid organ transplantation oraging or immunesenescence.

According yet further aspects of the present invention extend to themethods of administrating cytokinin compounds as detailed herein for thetreatment of the above-mentioned conditions.

A further aspect of the present invention provides the use of acytokinin compound in the preparation of a medicament for the preventionand/or treatment and/or amelioration of a blood cell deficiencydisorder, such as neutropenia, thrombocytopenia or anaemia in anindividual.

In one embodiment, the cytokinin compound is N6 isopentyl adenosine oran analogue or a pharmaceutically acceptable salt thereof. In a furtherembodiment the cytokinin compound is N6 benzyl adenosine or an analogueor pharmaceutically acceptable salt thereof.

In a further embodiment the cytokinin compounds is a prodrug of thecytokinins that can convert to the biologically active compound bymetabolism or hydrolysis.

A yet further aspect of the present invention provides a pharmaceuticalcomposition for the prevention and/or treatment and/or amelioration of ablood cell deficiency disorder, such as neutropenia, thrombocytopenia oranaemia in an individual the composition comprising a cytokinin compoundalong with at least one pharmaceutically acceptable diluent.

In one embodiment, the cytokinin compound is N6 isopentyl adenosine oran analogue or a pharmaceutically acceptable salt thereof. In a furtherembodiment the cytokinin compound is N6 benzyl adenosine or an analogueor pharmaceutically acceptable salt thereof.

In a further embodiment the cytokinin compounds is a prodrug of thecytokinins that can convert to thebiologically active compound bymetabolism or hydrolysis.

The inventor has further identified the surprising utility of oltiprazas well as analogues, derivatives, prodrugs and metabolites thereof inthe treatment and/or prophylaxis of blood cell number depletion andconditions such as immune dysregulation and cellular oxidative damage.Such conditions may result from exposure of a subject to radiation, suchas gamma radiation.

The compounds of the invention have shown, in irradiated mouse studies,that they have the ability to prevent and treat conditions associatedwith a depletion in blood cell number such as, but not limited to;neutropenia, thrombocytopenia, and anaemia. Without wishing to be boundby theory, the inventor theorises that said compounds are believed tocounteract and neutralize the oxidative damage which radiation causes inliving tissue.

In further embodiments, the compound may be 3H-1,2-dithiole-3-thione,anetol trithion and/or sulforaphane, and/or narigin.

In a further embodiment the compound may be selected from the groupcomprising 5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione, ADT, ADO,1,2-dithiole-3-thione, 1,2-dithiolane, 1,3-dithiole-2-thibne, andmalptilate.

In a further embodiment the compound chelates with, or forms a complexwith, one or more divalent or trivalent radioactive metal ions, wherebythe divalent or trivalent radioactive ions in the subject's cells ortissues are redistributed or sequestered such that the ions are limitedin their capacity to participate in unwanted tissue destruction.

In one embodiment the divalent or trivalent metal ions are selected fromFe, Cu, Ni, Ca, Mg, Mn, Cd, Pb, Al, Hg, Co, I, Se, Cs, U, Pa, Th, Ra,Ce, and Zn ions.

In a further embodiment a compound of Formula 2 enhances the cellularproduction of phase II detoxification enzymes following their depletionby radiation exposure The phase II detoxification enzymes may beselected from the group consisting of glutathione S transferase,gamma-glutamylcysteine synthetase, glutathione reductase, glutathioneperoxidase, epoxide hydrase, AFB-1 aldehyde reductase, glucuronylreductase; glucose-6-phosphate dehydrogenase, UDP-glucuronyl transferaseand AND(P)H:quinone oxidoreductase.

In further embodiments the Formula 2 compound as defined above andselected from the group consisting of:4-(3,5-diisopropyl-4-hydroxyphenyl)-1,2-dit-hiole-3-thione;4-(3,5-di-t-butyl-4-hydroxyphenyl)-1,2-dithiole-3-thione;4-[3,5-bis(l,l-dimethylpropyl)-4-hydroxyphenyl]-1,2-dithi-ole-3-thione;4-[3,5bis(l,l-dimethylbutyl)-4-hydroxyphenyl]-1,2-dithiole-3-thione;4-[3,5-bis(1,1,3,3-tetramethylbutyl)-4-hydrdxyphenyl]-1,2-dithole-3-thion-e;4-[3,5-bis(l-methylcyclohexyl)-4-hydroxyphenyl]-1,2-dithiole-3-thione;4-[3,5-bis(l,l-dimethylbenzyl)-4-hydroxyphenyl]-1,2-dithiole-3-thione;4-(3t-butyl-4-hydroxy-S-isopropylphenyl)-1,2-dithiole-3-thione;4-(3t-butyl-4-hydroxy-5-methylphenyl)-1,2-dithiole-3-thione;4-[3(1,1-dimethylpropyl)-4-hydroxy-5-isopropylphenyl]-1,2-dithiole-3-thi-one;4-[3(1,1-dimethylbenzyl)-4-hydroxy-5-isopropylphenyl]-1,2-dithole-3-thione;5-benzylthio-4-(3,5-di-tbutyl-4-hydroxyphenyl)-1,2-dithole-3-thion-e;5-benzylthio-4-[3,5-bis(l,l-dimethylpropyl)-4-hydroxy-phenyl]-1,2-dithi-ole-3-thione;5-hexylthio-4-(3,5-di-t-butyl-4-hydroxyphenyl)-1,2-dithole-3-thione;5-hexylthio-4-[3,5bis(l,l-dimethylbutyl)-4-hydroxy-phenyl]-1,2-d-thione-3-thione;5-octadecylthio-4-(3,5-di-t-butyl-4-hydroxyphenyl)-1,2-di-thiole-3-thione;5-octadecylthio-4-[3,5-bis(l,l-dimethylbenzyl)-4-hydroxyp-henyl]-1,2-dithiole-3-thione;5-allylthio-4-(3,5-di-t-butyl-4-hydroxypheny-l)-1,2-dithiole-3-thione;5-cyclohexylthio-4-(3,5-di-t-butyl-4-hydroxyphenyl)-1,2-dithiole-3-thione;and 4-(3,5-di-sec-butyl-4-hydroxyphenyl)-1,2-di-thiole-3-thione.

In one embodiment, the method further has utility in the treatment ofsymptoms of neutropenia, thrombocytopenia or anaemia, and accordinglyOltipraz or derivatives or analogues thereof may be administered toindividuals in order to treat such conditions.

In one embodiment, the neutropenia is postinfectious neutropenia,autoimmune neutropenia, chronic idiopathic neutropenia or a neutropeniaresulting from or potentially resulting from a cancer chemotherapy,chemotherapy for an autoimmune disease, an antiviral therapy, directradiation exposure, secondary radiation exposure through environmentalcontamination, tissue or solid organ allograft or xenograft rejection orimmune suppression therapy in tissue or solid organ transplantation oraging or immunesenescence.

According yet further aspects of the present invention extend to themethods of administrating oltipraz as detailed herein for the treatmentof the above-mentioned conditions.

A further aspect of the present invention provides the use of oltiprazor a derivative or analogue thereof in the preparation of a medicamentfor the prevention and/or treatment and/or amelioration of a blood celldeficiency disorder, such as neutropenia, thrombocytopenia or anaemia inan individual.

A yet further aspect of the present invention provides a pharmaceuticalcomposition for the prevention and/or treatment and/or amelioration of ablood cell deficiency disorder, such as neutropenia, thrombocytopenia oranaemia in an individual the composition comprising Oltipraz or aderivative or analogue thereof along with at least one pharmaceuticallyacceptable diluent.

The present inventor has further identified that administering atherapeutic which is a combination of a cytokinin compound along witholtipraz or a derivative or analogue thereof results in a compositionwhich exhibit a synergistic benefit over the administration of eithercompound alone in relation to the prevention and/or treatment and/oramelioration of a blood cell deficiency disorder, such as neutropenia,thrombocytopenia or anaemia in an individual.

Accordingly a yet aspect of the provides a method for the preventionand/or treatment and/or amelioration of a blood cell deficiencydisorder, such as neutropenia, thrombocytopenia or anaemia in anindividual, the method comprising the step of administering to anindividual in need of such treatment a therapeutically effective amountof a cytokinin compound along with Itipraz or an analogue or derivativethereof.

In one embodiment, the cytokinin compound is N6 isopentyl adenosine oran analogue or a pharmaceutically acceptable salt thereof. In a furtherembodiment the cytokinin compound is N6 benzyl adenosine or an analogueor pharmaceutically acceptable salt thereof.

In a further embodiment the cytokinin compounds is a prodrug of thecytokinins that can convert to the biologically active compound bymetabolism or hydrolysis.

In one embodiment the individual in need of treatment with the method ofthis aspect of the invention is a human who has a blood cell deficiencydisorder.

Where a patient is treatment in accordance with the method of thisaspect of the present invention, it is preferred that the treatmentreduces (1) the severity of pain during vascular or microvascularocclusions, (2) the severity of vascular or microvascular occlusions or(3) the frequency of vascular or microvascular occlusions.

A further aspect of the present invention provides the use of a combinedmedicament, said medicament comprising at least one cytokinin compoundand oltipraz in the preparation of a medicament for the preventionand/or treatment and/or amelioration of a blood cell deficiencydisorder, such as neutropenia, thrombocytopenia or anaemia in anindividual.

In a further embodiment the cytokinin compounds is a prodrug of thecytokinins that can convert to the biologically active compound bymetabolism or hydrolysis.

A yet further aspect of the present invention provides a combinedpharmaceutical composition for the prevention and/or treatment and/oramelioration of a blood cell deficiency disorder, such as neutropenia,thrombocytopenia or anaemia in an individual the composition comprisinga cytokinin compound, and oltipraz along with at least onepharmaceutically acceptable diluent.

In one embodiment, the cytokinin compound is N6 isopentyl adenosine oran analogue or a pharmaceutically acceptable salt thereof. In a furtherembodiment the cytokinin compound is N6 benzyl adenosine or an analogueor pharmaceutically acceptable salt thereof.

In a further embodiment the cytokinin compounds is a prodrug of thecytokinins that can convert to the biologically active compound bymetabolism or hydrolysis.

A yet further aspect of the present invention provides a combinedmedicament comprising Formula 1 and Formula 2 compounds administered toan individual in need of therapy for the prevention and/or treatmentand/or amelioration of a blood cell deficiency disorder, such asneutropenia, thrombocytopenia or anaemia. Such compounds may be referredto herein as Formula 3 compounds “F3cs”.

In one embodiment the treatment comprises administering said formulationof Formula 1 alone or in combination with Formula 2 compounds in unitdoses of 0.01 mg. to 1000 mg/per Kg of bodyweight.

In a further embodiment the compound of Formula 1 and/or Formula 2 aremicronized or the compounds are present in a composition that comprisesa pharmaceutically acceptable carrier, the carrier optionally selectedfrom phosphatidylcholine, diphosphatidylcholine, vitamin E, acyclbdextrih, magnolol, a microbial preservative, water or a liquidexcipient suitable for ophthalmic pharmaceutical formulations, orformulated in a tissue decomposition matrix to allow slow delivery froma polymeric delivery such as with the use of silicone polymers.

It is preferable that the pharmaceutical composition of this aspect ofthe present invention is provided by combining independently formulateddrugs, or by preparing a combination formulation consisting of a mixtureof drugs. When the pharmaceutical composition of the present inventionis to be used in actuality, unit dosage forms suitable for oraladministration are to be formulated and administered according to theconventions of the proper pharmaceutical field. To achieve this, theoral formulation comprises a hard or soft capsule, tablet, powder, etc.The oral formulation, in addition to oltipraz/cytokinin compounds as thepharmacologically active agent, may contain one or morepharmacologically non-active conventional carrier mediums. For examplethe oral formulation may contain as additives starch, lactose,carboxymethylcellulose, kaolin, and the like excipients; water, gelatin,alcohol, glucose, arabic gum, tragacanth gum and the like binders;starch, dextrine, sodium alginate, and the like disintegrants; talc,stearic acid, magnesium stearate, liquid paraffin, and the likelubricants. Dissolving aids may be further added.

The daily dosage of the present invention depends on various factorssuch as the patient's degree of liver damage, time of onset ofhepatitis, age, health, complications, etc. However, for the averageadult, the oltipraz/cytokinin compounds composition is administered onceor twice a day for a total daily dosage of 5 to 200 mg, more preferably25 to 50 mg. However, in patients with severe liver damage or when usedas an anti-recurring agent after hepatic carcinectomy, the presentinvention can depart from the scope of the above pharmaceuticalcomposition and employ even large dosages. Most preferably, one or twounit dosages containing 25 mg of oltipraz and 5 mg of cytokinincompounds are orally administered twice a day.

Modulation of DNA Repair Mechanisms After Radiation Exposure DNA Damage

DNA damage, due to normal metabolic processes inside the cell, occurs ata rate of 50,000 to 500,000 molecular lesions per cell per day.

DNA damage can be subdivided into two main types:

endogenous damage such as attack by reactive oxygen radicals producedfrom normal metabolic by products (spontaneous mutation);

(i) exogenous damage caused by external agents such as ultraviolet [UV200-300 nm] radiation from the sun, (ii) other radiation frequencies,including x-rays and gamma rays, (iii) hydrolysis or thermal disruption

(iv) certain plant toxins, (iv) human-made mutagenic chemicals, such ashydrocarbons from, cigarette smoke, and (v) cancer chemotherapy andradiotherapy.

Before cell division the replication of damaged DNA can lead to theincorporation of wrong bases opposite damaged ones. After the wrongbases are inherited by daughter cells these become mutated cells (cellsthat carry mutations), and there is no way back (except through the rareprocesses of back mutation and gene conversion).

DNA Repair Mechanisms

Cells cannot tolerate DNA damage that compromises the integrity andaccessibility of essential information in the genome (but cells remainsuperficially functional when so-called “non-essential” genes aremissing or damaged). Depending on the type of damage inflicted on theDNA's double helical structure, a variety of repair strategies hasevolved to restore lost information. As templates for restoration cellsuse the unmodified complementary strand of the DNA or the sisterchromosome. Without access to template information, DNA repair iserror-prone (but this can be the standard pathway, e.g. most doublestrand-breaks in mammalian cells are repaired without templateassistance; see below).

Damage to DNA alters the spatial configuration of the helix and suchalterations can be detected by the cell. Once damage is localized,specific DNA repair molecules are summoned to, and bind at or near thesite of damage, inducing other molecules to bind and form a complex thatenables the actual repair to take place. The types of molecules involvedand the mechanism of repair that is mobilized depend on the type of DNAdamage at stake arid whether the cell has entered into a state ofsenescence the phase of the cell cycle that the cell is in.

Single Strand and Double Strand DNA Damage

When only one of the two strands of a chromosome has a defect, the otherstrand can be used as a template to guide the correction of the damagedstrand. In order to repair damage to one of the two helical domains ofDNA, there are numerous mechanisms by which DNA repair can take place.These include direct reversal of damage by various mechanisms thatspecialize in reversing specific types of damage. Examples includemethyl guanine methyl transferase (MGMT) which specifically removesmethyl groups from guanine, and photolyase in bacteria, which breaks thechemical bond created by UV light between adjacent thymidine bases. Notemplate strand is required for this form of repair.

Excision repair mechanisms that remove the damaged nucleotide replacingit with an undamaged nucleotide complementary to the nucleotide in theundamaged DNA strand. These include Base excision repair (BER), whichrepairs damage due to a single nucleotide caused by oxidation,alkylation, hydrolysis, or deamination; Nucleotide excision repair(NER), which repairs damage affecting 2-30 nucleotide-length strands.These include bulky, helix distorting damage, such as thyminedimerization caused by UV light as well as single-strand breaks. Aspecialized form of NER known as Transcription-Coupled Repair (TCR)deploys high-priority NER repair enzymes to genes that are beingactively transcribed; Mismatch repair (MMR), which corrects errors ofDNA replication and recombination that result in mispaired nucleotidesfollowing DNA replication.

Double Strand Breaks

A particularly hazardous type of DNA damage to dividing cells is a breakto both strands in the double helix. Two mechanisms exist to repair thisdamage. They are generally known as Non-Homologous End-Joining andrecombinational repair, template-assisted repair, or homologousrecombination.

Recombinational repair requires the presence of an identical or nearlyidentical sequence to be used as a template for repair of the break. Theenzymatic machinery responsible for this repair process is nearlyidentical to the machinery responsible for chromosomal crossover in germcells during meiosis. The recombinational repair mechanism ispredominantly used during the phases of the cell cycle when the DNA isreplicating or has completed replicating its DNA. This allows a damagedchromosome to be repaired using the newly created sister chromatid as atemplate, i.e. an identical copy that is moreover orderly paired to thedamaged region. Many genes in the human genome are present in multiplecopies providing many possible sources of identical sequences. Butrecombinational repair that relies on these copies as templates for eachother is problematic because if leads to chromosomal translocations andother types of chromosomal rearrangements. Non-Homologous End-Joining(NHEJ) rejoins the two ends of the break in absence of a templatesequence. However there is often DNA sequence loss during this processand so this repair can be mutagenic. NHEJ can occur at all stages of thecell cycle but in mammalian cells is the main repair mechanism until DNAreplication makes it possible for recombinational repair to use thesister chromatid as a template. Since the vast majority of the genome inhumans and other multicellular organisms is made up of DNA that containsno genes, the so-called “junk DNA”, mutagenic NHEJ is likely to be lessharmful than template-assisted repair would be in presence of multipletemplate sequences, since in the latter case undesirable chromosomalrearrangements are generated. The enzymatic machinery used for NHEJ isalso utilized in B-cells to rejoin breaks created by the RAG proteinsduring VDJ recombination a crucial step in the generation of antibodydiversity by the immune system.

Procedures such as chemotherapy and radiotherapy work by overwhelmingthe capacity of the cell to repair DNA damage and resulting in celldeath. Cells that are most rapidly dividing such as cancer cells arepreferentially affected. The side effect is that other non-cancerous butsimilarly rapidly dividing cells such as stem cells in the bone marroware also affected. Modern cancer treatments attempt to localize the DNAdamage to cells and tissues only associated with cancer.

The inventors have surprisingly found that oltipraz can induce DNArepair mechanisms. Such DNA repair can, in particular be mediatedfollowing radiation exposure.

Accordingly a yet further aspect of the present invention provides amethod of mediating DNA repair in a cell following radiation damagewhich requires such repair, the method comprising the steps of bringinga therapeutically effective amount of oltipraz or a derivative oranalogue thereof into contact with the cell.

In one embodiment the oltipraz derivative or analogue thereof isselected from the group comprising 3H-1dithiole-S-thione, sulforaphane,5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione, ADT, ADO,1,2-dithiole-3-thione, 1,2-dithiolane, 1,3-dithiole-2-thione, andmalotilate.

In one embodiment, the cell which requires DNA repair followingradiation damage is located in an individual and accordingly this aspectof the invention further extends to a method of treating an individualfor DNA repair, the method comprising the steps of administering to theindividual a therapeutically effective amount of oltipraz.

In a further embodiment there is provided the use of oltipraz or aderivative or analogue thereof in the preparation of a medicament forthe stimulation of cellular DNA repair mechanisms following radiationexposure.

In one embodiment the oltipraz derivative or analogue thereof isselected from the group comprising 3H-1,2-dithiole-3-thione,sulforaphane, 5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione, ADT, ADO,1,2-dithiole-3-thione, 1,2-dithiolane, 1,3-dithiole-2-thione, andmalotilate.

A yet further aspect of the present invention provides a pharmaceuticalcomposition for mediating DNA repair following radiation exposurecomprising oltipraz or a derivative or analogue thereof along with apharmaceutically acceptable carrier.

In one embodiment the oltipraz derivative or analogue thereof isselected from the group comprising 3H-1,2-dithiole-3-thione,sulforaphane, 5-(4-methdxyphenyl)-3H-1,2-dithiole-3-thione, ADT, ADO,1,2dithiole-3-thione, 1,2-dithiolane, 1,3-dithiole-2-thione, andmalotilate.

Immunosuppression Caused by Space Flight

The conditions experienced during space flight, such as isolation,stress, containment, microgravity and radiation are all thought tomediate a suppressive effect on the immune system. In particular,radiation can cause damage to human bone marrow stem cells which are theprecursor cells for the cells of the immune system.

Human exposure to solar radiation during space travel can amount to upto 3 Gy of proton and gamma radiation. This can result inimmunosuppression which can further lead to the reactivation of latentviral infection and malignancy. The immune cells which are particularlysusceptible to such solar radiation include; bone marrow stem cells(CD34 cells), helper T cells (CD4+), cytotoxic T cells (CD8+), B cells(CD19+), monocytes and macrophages (CD19+) and natural killer (NK) cells(CD56+).

The present inventor has identified that the compounds of the presentinvention have further utility in the treatment and prevention ofradiation induced immunosuppression during space travel.

Accordingly a further aspect of the present invention provides a methodof treating and or preventing radiation induced immunosuppression,wherein the radiation is derived from solar radiation, particularlyproton or gamma radiation, the method comprising administering to anindividual in need of such treatment a therapeutically effective amountof at least one of the compounds of the present invention.

Treatment/Therapy

The term ‘treatment’ is used herein to refer to any regimen that canbenefit a human or non-human animal. The treatment may be in respect ofan existing condition or may be prophylactic (preventative treatment).Treatment may include curative, alleviation or prophylactic effects.

More specifically, reference herein to “therapeutic” and “prophylactic”treatment is to be considered in its broadest context. The term“therapeutic” does not necessarily imply that a subject is treated untiltotal recovery. Similarly, “prophylactic” does not necessarily mean thatthe subject will not eventually contract a disease condition.

Accordingly, therapeutic and prophylactic treatment includesamelioration of the symptoms of a particular condition or preventing orotherwise reducing the risk of developing a particular condition. Theterm “prophylactic” may be considered as reducing the severity or theonset of a particular condition. “Therapeutic” may also reduce theseverity of an existing condition.

As used herein, the term “therapeutically effective amount” means theamount of a composition which is required to reduce the severity ofand/or ameliorate blood cell depletion or at least one condition orsymptom which results therefrom.

As used herein, the term “prophylactically effective amount” relates tothe amount of a composition which is required to prevent the initialonset, progression or recurrence of blood cell depletion or at least onesymptom or condition which results from blood cell depletion by theadministration of the compounds of the present invention.

As used herein, the term “subject” refers to an animal, preferably amammal and in particular a human. In a particular embodiment, thesubject is a mammal, in particular a human, who has been, or who isgoing to be exposed to radiation, for example radiation therapy such aschemotherapy or radiotherapy. The term “subject” is interchangeable withthe term “patient” as used herein.

Administration

The products of the invention may be administered alone but willpreferably be administered as part of a pharmaceutical composition,which will generally also comprise a suitable pharmaceutical exeipient,diluent or carrier which would be selected depending on the intendedroute of administration.

The products of the invention may be administered to a patient in needof treatment via any suitable route. The precise dose will depend upon anumber of factors.

Route of administration may include; parenterally (includingsubcutaneous, intramuscular, intravenous, by means of, for example adrip patch), some further suitable routes of administration include (butare not limited to) oral, rectal, nasal, topical (including buccal andsublingual), infusion, vaginal, intradermal, intraperitoneally,intracranially, intrathecal and epidural administration oradministration via oral or nasal inhalation, by means of, for example anebuliser or inhaler, or by an implant.

In preferred embodiments, the composition is administered orally, or isadministered to the lungs as an aerosol via oral or nasal inhalation.

For administration via the oral or nasal inhalation routes, preferablythe active ingredient will be in a suitable pharmaceutical formulationand may be delivered using a mechanical form including, but notrestricted to an inhaler or nebuliser device.

Further, where the oral or nasalinhalation routes are used,administration is by a SPAG (small particulate aerosol generator) may beused.

For intravenous injection, the active ingredient will be in the form ofa parenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as sodium chloride injection, Ringer's injection,Lactated Ringer's injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally comprise a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

The composition may also be administered via microspheres, liposomes,other microparticulate delivery systems or sustained releaseformulations placed in certain tissues including blood. Suitableexamples of sustained release carriers include semipermeable polymermatrices in the form of shared articles, e.g. suppositories ormicrocapsules. Implantable or microcapsular sustained release matricesinclude polylactides (U.S. Pat. No. 3,773,919; EP-A-0058481) copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al,Biopplymers 22(1): 547-556, 1985), poly (2-hydroxyethyl-methacrylate) orethylene vinyl acetate (Langer et al, J. Biomed. Mater. Res. 15:167-277, 1981, and Langer, Chem. Tech. 12:98-105, 1982).

Examples of the techniques and protocols mentioned above and othertechniques and protocols which may be used in accordance with theinvention can be found in Remington's Pharmaceutical Sciences, 18thedition, Gennaro, A. R., Lippincott Williams & Wilkins; 20th edition(Dec. 15, 2000) ISBN 0-912734-04-3 and Pharmaceutical Dosage Forms andDrug Delivery Systems; Ansel, H. C. et al. 7th Edition ISBN0-683305-72-7 the entire disclosures of which is herein incorporated byreference.

Pharmaceutical Compositions

The actual amount administered, and rate and time-course ofadministration, will depend on the nature and severity of what is beingtreated. Prescription of treatment, e.g. decisions on dosage etc, isultimately within the responsibility and at the discretion of generalpractitioners and other medical doctors, and typically takes account ofthe radiation damage to be treated, the condition of the individualpatient, the site of delivery, the method of administration and otherfactors known to practitioners.

Prescription of treatment, e.g. decisions on dosage etc, is ultimatelywithin the responsibility and at the discretion of generalpractitioners, physicians or other medical doctors, and typically takesaccount of the disorder to be treated, the condition of the individualpatient, the site of delivery, the method of administration and otherfactors known to practitioners.

The optimal dose can be determined by physicians based on a number ofparameters including, for example, age, sex, weight, severity of thecondition being treated, the active ingredient being administered andthe route of administration.

Unless otherwise defined, all technical and scientific terms used hereinhave the meaning commonly understood by a person who is skilled in theart in the field of the present invention.

Throughout the specification, unless the context demands otherwise, theterms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or‘comprising’, ‘includes’ or ‘including’ will be understood to imply theinclusion of a stated integer or group of integers, but not theexclusion of any other integer or group of integers.

As used herein, terms such as “a”, “an” and “the” include singular andplural referents unless the context clearly demands otherwise. Thus, forexample, reference to “an active agent” or “a pharmacologically activeagent” includes a single active agent as well a two or more differentactive agents in combination, while references to “a carrier” includesmixtures of two or more carriers as well as a single carrier, and thelike.

DETAILED DESCRIPTION OF THE INVENTION Salts and Solvates

The active compounds disclosed herein can, as noted above, can beprepared in the form of their pharmaceutically acceptable salts.Pharmaceutically acceptable salts are salts that retain the desiredbiological activity of the parent compound and do not impart undesiredtoxicological effects. Examples of pharmaceutically acceptable salts arediscussed in Berge et al., 1977, “Pharmaceutically Acceptable Salts,” J.Pharm. ScL, Vol. 66, pp. 1-19.

The active compounds disclosed may also be prepared in the form of theirsolvates. The term “solvate” is used herein in the-conventional sense torefer to a complex of solute (e.g., active compound, salt of activecompound) and solvent. If the solvent is water, the solvate may beconveniently referred to as a hydrate, for example, a hemihydrate,monohydrate, dihydrate, trihydrate, tetrahydrate, and the like.

Prodrugs

The invention further extends to prodrugs of the compounds of thepresent invention. A prodrug of any of the compounds can be made usingwell known pharmacological techniques.

Homoloques and Analogues

The present invention is further intended to encompass, in addition tothe use of the above listed compounds, the use of homologues,derivatives and analogues of such compounds. In this context, homologuesare molecules having substantial structural similarities to theabove-described compounds and analogues are molecules having substantialbiological similarities regardless of structural similarities.

The invention further provides kits for carrying but the therapeuticregimens of the invention. Such kits may comprise, in one or morecontainers, therapeutically or prophylactically effective amounts of thecompositions of the invention in a pharmaceutically acceptable form.Such kits may further include instructions for the use of thecompositions of the invention, or for the performance of the methods ofthe invention, or may provide further information to provide a physicianwith information appropriate to treating blood cell depletion orconditions resulting therefrom.

The present invention will now be described with reference to thefollowing examples which are provided for the purpose of illustrationand are not intended to be construed as being limiting on the presentinvention. The present invention will make reference to the followingfigures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a graph illustrating B lymphocyte function followingadministration of IPA, and

FIG. 2 shows a graph illustrating T lymphocyte function followingadministration of IPA.

EXAMPLES Example 1 Materials and Methods:

Riboflavin, Nitroblue tetrazolium (NBT), reduced glutathione (GSH), S-S′dithiobis (2-nitrobenzdic acid) (DTNB), and 1-chloro-2,4-dinitrobenzene(CDNB) were obtained from Sisco Research Laboratories Pvt. Ltd, Mumbai,India. Thiobarbituric acid was purchased from Hi-media laboratories,Mumbai, India, 1,1,3,3-tetramethoxy propane was supplied fromSigma-Aldrich USA. All other chemicals and reagents used in this studywere of analytical grade. Oltipraz was supplied by the contractorCanopus Corp.

Inbred 4-6 weeks old female Swiss albino mice (20-30 g) were obtainedfrom Small animal breeding station, Kerala Agricultural University,Mannuthy, Thrissur. Animals were kept in well-ventilated cages understandard conditions of temperature, pressure and humidity. The animalswere provided with normal mouse chow and water ad libitum. All animalexperiments conducted during the present study got prior permission andfollowed the guidelines of Institutional Animal Ethics Committee (IAEC).

Irradiation

Animals were treated with a single dose of radiation of 700 rads (7Gy).The source of radiation was a 6° Co Theratron-Phoenix teletherapy unit(Atomic Energy Ltd, Canada). Animals were restrained in speciallydesigned, well-ventilated cages without anesthesia and exposed to wholebody radiation at a rate of 1.33 Gy/min in a field size of 25×25 cm² andat a distance of 80 cm from the source.

Determination of effect of oltipraz on haematological parameters ofirradiated animals.

Twenty-four mice were randomly divided into three groups of 8 animalseach. Group I was treated as irradiated control served with vehicle.Group II was treated with oltipraz (50 mgs/Kg b wt) ten days prior toirradiation. Group III was administered oltipraz (50 mgs/Kg b. wt) tendays prior to irradiation and continued for another fifteen days afterirradiation. All the three groups were irradiated with a single dose of750 rads.

Body Weights of all the animals were determined one day prior toirradiation and every third day thereafter. Bipod was collected fromtail vein into heparinized tubes and the following parameters wereanalyzed one day before radiation and every third day thereafter. Theparameters analyzed were total WBC count (haembcytometer method),differential count (Leishman's staining method) and haemoglobin byDrabkin's method). Determination of effect of oltipraz on bone marrowviability arid antioxidant parameters of irradiated animals.

Thirty six animals were divided into four groups of nine animals each.For group 1 to 4 treatment protocol were similar as described above.Group 4 was treated as normal animals without any treatment. On days 5,10 and 15 after irradiation (750) rads three animals from each group wassacrificed. Blood was collected to heparinized tubes, and plasma wasremoved and following parameters were assayed in the blood. Activity ofthe enzyme SOD was measured by NBT reduction method of McGord andFridovich. CAT activity was estimated by the method of Aebi by measuringthe rate of decomposition of hydrogen peroxide at 240 nm. Level of GSHwas assayed by the method of Moron et al based on the reaction withDTBN. Assay of GPX followed the method of Hafeman based on thedegradation of H₂O₂ in the presence of GSH. The method of Habig wasfollowed to assay the activity of GST based on the rate of increase inthe conjugate formation between GSH and CDNB.

The femurs of three above animals were dissected out and bone marrowcells were flushed into phosphate buffered saline (pH 7.4) containing 2%foetal calf serum. The cells were washed and bone marrow viability wasdetermined by the method of Sredni. The results were expressed as numberof live bone marrow cells ×10(6)/femur.

The liver of the sacrificed animals were excised quickly washed inice-cold saline and kept at −70° C. till the day of analysis. On the dayof analysis 25% homogenate was pre-pared in tris-HCl buffer (0.1 M,pH7.4). The homogenate was centrifuged at 12000 rpm for 30 minutes andsupernatant was used to determine the tissue lipid peroxide levels (LPO)using the TBA method of Okhawa et al.

Data Analysis

Data was expressed as mean±standard deviation (SD). Significance levelsfor comparison of differences were deter-mined using Student's t test.The mean of Oltipraz treated group was compared with that of radiationalone treated group. The radiation alone treated group was then furthercompared with untreated group. The differences between means wereconsidered to be statistically significant if p<−0.001.

Radiation treatment at the dose level used here did not produce astatistically significant reduction in the body-weight of the exposedanimals. Initial body weight of animals were 26.27±3.76, 23.92±4.61 and24.98±3.76 respectively for group I, II and III. On day 6 body weightwas reduced to 21.82±3.34, 21.02±4.14 and 21.10±2.51 respectively(p>0.05). Radiation significantly lowered the total leukocyte count inirradiated animals. Administration of oltipraz was found to increase thecount. In the initial days after irradiation both group II (oltipraz pretreated group) and group III (oltipraz continuously administered group)showed almost similar number of WBC. But at later days afterirradiation, group III showed a significantly elevated WBC as comparedwith group I (radiation alone treated group) and group II. Thisindicated that continuous oltipraz administration stimulated thehaematopoietic system in a concentration dependent manner.

This observation is further supported by the increased bone marrowviability found in-group III. Bone marrow viability in normal animalswas (Group IV) was 16.21±0.45×10⁶ cells/femur. Bone marrow viability wassignificantly decreased after irradiation. After the 15th day ofpost-irradiation group II possessed a value of 6.1×10⁶ cells/femur whereas group II and III showed 5.82×106 and 14.32×10⁶ cells/femurrespectively. The haemoglobin levels were significantly reduced afterirradiation. On day 6 radiation alone treated group had a hemoglobinlevel of 10.37±3.19 where as oltipraz continuously administered grouphad a value of 12.42±2.76. The differential count did not show anysignificant variation.

The activity of both SOD and CAT, two of the major enzymes involved inthe antioxidant defence mechanism were found to be decreased afterirradiation The continuous administration of Oltipraz enhanced SODactivity, which showed the maximum value on the 15th day afterirradiation and CAT on tenth day after irradiation.

Activity of GPX was also found to be decreased after whole bodyirradiation. Continuous administration of oltipraz elevated the activityof GPX. On the 15th day after irradiation group I had an activity of1254.00±116.23 U/L of haemolysate whereas group III showed an activityof 1927.87±136.06 demonstrating that oltipraz administration stimulatedGPX activity (p<−0.001). The levels of the major cellular antioxidantGSH increased after oltipraz administration. The levels of GSH werebrought down after irradiation. On the 15th day the levels of GSHincreased almost three times in oltipraz continuously administered groupas compared with radiation alone treated group indicating that oltiprazadministration elevated the GSH levels (p<0.001), It could be presumedthat an increased level of anti-oxidant enzymes and GSH is a directconsequence of oltipraz administration and could be seen inun-irradiated animals as well. Oltipraz administration also elevated theactivity of GST, an enzyme involved in the glutathione mediateddetoxification system. On the 15th day group III showed an activity of2.38±0.25 (nano-moles of CDNB-GSH conjugate formed) (p<−0.01) where asgroup I had an activity of 1.51±0.37 only. Radiation increased thelevels of lipid peroxidation in all the radiation treated animals. Onday 15, it was 3.47±0.31 (nano-moles of MDA formed/mg of protein forgroup I, whereas in-group III it was significantly reduced to a level of2.12±0.21 (p<−0.001).

Example 2 Patient Case Study

The patient was treated with a Formula 2 compound and blood cellparameters were monitored. The sample size for this experiment was 1,consisting of subject 1. Subject 1 is an immune comprised patient withmultiple recurrent infectious bouts requiring hospitalization while in anearly moribund condition began treatment with the experimental drugIsopentyl adenosine (IPA).

The subject had been hospitalized with avert life-threateninginfections. The subject's total white blood cell counts were in therange of 2200-4900 for the three month period preceding hospitalization.A T4/T8 cell ratio of 50/270 (0.22) was observed two months earlier. Thepatient was hospitalized from depression, exhaustion, bronchialinfection, severe diarrhoea, severe weight loss and complete loss ofappetite and spiked fever. The patient's weight was 98 pounds.

The patient, although initially was very co-operative relative toreceiving experimental IPA therapy, for no rational reasons at varioustimes stopped IPA.

Formula 2 Isopentvl Adenosine (IPA) Therapy—Immunological Studies

The subject initially received IPA (see FIGS. 1 and 2). T and Blymphocyte competence as measured by the ability of these cells toproliferate in response to specific stimulating mitogens (PHA for Tcells and PWM for B cells) which was monitored on a weekly basis untildeath. As shown in FIGS. 1 and 2 respectively the subject's T and Blymphocyte competence on was well below normal. Indeed, the competenceof both these immune cell populations was less than the 1st percentileof normal donors; that is, greater than 99% of all normal donorshistorically tested (>1460 normal donors in our laboratory) hadresponses greater than the patient demonstrated.

A major rapid recovery of both T and B lymphocyte competence wasexperienced within less than 7 days after initiation of IPA therapy. Thelevels of competence within 7 days of drug therapy initiation were abovethe lower iimitsrof the normal donor's reactivity. This represented aremarkable recovery since patients receiving. Most immune stimulatingagents (e.g., BCG, Leyamisole) exhibit a slower and less dramatic immunerecovery capacity. The subject conscientiously continued to takeconstant oral doses of IPA.

The subject felt cured and abruptly stopped taking the drug. No oneexcept the subject and his friend were aware of this unilateral decisionuntil several weeks later. Fever and diarrhoea in the subject hadcompletely subsided and the patient exhibited a 15 pound weight gainduring this time.

As shown in FIGS. 1 and 2, at the time that the patient proclaimedself-cure and thus stopped all IPA medication, his T and B lymphocytecompetence again dropped to less than 1% or normal donors' competence.

This was most dramatically observed with T lymphocyte function but wasalso observed in somewhat lagged fashion with B lymphocyte function.Total WBC counts were in the range of 1650 to 2550 during the period.His peripheral blood showed 50 T4 cells and 220 T8 cells (0.23 ratio).

The patient stopped Formula 2 IPA therapy for a few days. During thistime both T and B lymphocyte competence rapidly bottomed out, thepatient was hospitalized in for a 7 to 10 day period for severebronchial infections and fever, diarrhoea, anxiety and weight loss.

The subject resumed Formula 2 IPA therapy at recommended dose levels.Minor T lymphocyte competence returned during the following week; Blymphocyte competence continued to decrease. Within two weeks, Tlymphocyte competence was again nearly within the normal range. AlthoughB lymphocyte competence was still somewhat depressed, this cell'scompetence was also improving. Within a month T lymphocyte competencehad risen to well within the range of normal donor reactivity, while Blymphocyte competence was somewhat below lower normal limits.

The subject's personal status demonstrated a dramatic improvement; hewas periodically dining out with a large appetite, diarrhoea hadlessened, fever had subsided, he was riding his bicycle daily andpartaking in physical activities. The subject again stopped takingmedication for a week without the physician's knowledge. T lymphocytecompetence again demonstrated a major sudden decrease to less than 1% ofnormal donors' activity.

The patient again exhibited spiked fever, acute diarrhoea andexhaustion. The subject again resumed Formula 2 IPA therapy thefollowing week, for one week, with one-half the recommended dosagetaken. The subject's T lymphocyte competence spiked dramatically toabove the lower limit of the normal range. B lymphocyte activelydemonstrated a slight rise. During the next week the dosage of IPA wasgreatly (>3×) increased. T lymphocyte competence demonstrated depressionsuggesting immunotoxicity by IPA; since a 33% decreased dosage thefollowing week resulted in T lymphocyte competence increasing onceagain.

The patient stopped, therapy during the next month, both T and Blymphocyte function fell to nil, as a direct consequence. The patientdied approximately two weeks later suffering from acute bronchialinfection accompanied with high fever, diarrhoea, exhaustion and weightloss.

In conclusion, direct correlations were observed during the period ofadministration of Formula 2 IPA with quality of life and depressed orelevated T and B lymphocyte function. Enhanced T and B lymphocytecompetence correlated well with continued Formula 2 IPA medication;optimum levels of competence appeared to be experienced at optimum IPAdose levels administered. Since competent T and B lymphocyte function isrequired for combating microbial infections by the host, majorinfectious bouts correlated with times when competence was most severelycompromised. The patient experienced 5 months of life with periods ofexcellent quality after initiation of Formula 2 IPA therapy at a timewhen he was terminally moribund. It would appear that the patient wouldhave enjoyed appreciable therapeutic benefits from Formula 2 IPA, if hehad conscientiously and continuously received IPA medication. If optimumdose levels had been more clearly established this would have presumablyoptimized quality and extension of life to a greater extend than wasactually observed in this patient.

All documents referred to in this specification are herein incorporatedby reference. Various modifications and variations to the describedembodiments of the inventions will be apparent to those skilled in theart without departing from the scope of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications Of the described modes of carrying Out theinvention which are obvious to those skilled in the art are intended tobe covered by the present invention. Reference to any prior art in thisspecification is not, and should not be taken as, an acknowledgment orany form of suggestion that this prior art forms part of the commongeneral knowledge in any country.

1. A method for the treatment and/or prophylaxis of blood celldepletion, the method comprising: providing a therapeutically effectiveor prophylactically effective amount of oltipraz or a derivative,analogue, metabolite, prodrug or pharmaceutically acceptable saltthereof; and administering the same to a subject in need thereof.
 2. Themethod as claimed in claim 1 wherein the blood cell depletion results inat least one condition selected from the group consisting ofneutropenia, thrombocytopenia, lymphocytopenia and anaemia.
 3. Themethod as claimed in claim 1 wherein the oltipraz metabolite ismetabolite
 3. 4. The method as claimed in claim 1 wherein oltipraz orthe derivative, analogue, metabolite, prodrug or pharmaceuticallyacceptable salt thereof is formulated with carboxymethyl cellulose toform a combined medicament.
 5. A pharmaceutical composition for use inthe treatment of blood cell depletion, the composition comprisingoltipraz or a derivative, analogue, metabolite, prodrug orpharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable diluent and/or carrier.
 6. The pharmaceutical composition asclaimed in claim 5 wherein oltipraz is provided in the form ofmetabolite
 3. 7. The pharmaceutical composition as claimed in claim 5wherein the composition is formulated with carboxymethyl cellulose.
 8. Amethod for the treatment and/or prophylaxis of blood cell depletion, themethod comprising: providing a therapeutically or prophylacticallyeffective amount of at least one cytokinin compound; and administeringthe same to a subject in need thereof.
 9. The method as claimed in claim8 further comprising the step of administering to the subject atherapeutically effective or prophylactically effective amount ofoltipraz or a derivative, analogue, metabolite, prodrug orpharmaceutically acceptable salt thereof
 10. The method as claimed inclaim 8 wherein the cytokinin compound is N⁶ isopentenyl adenosine or aderivative, analogue, metabolite, prodrug or pharmaceutically acceptablesalt thereof.
 11. The method as claimed in claim 10 wherein the N⁶isopentenyl adenosine or derivative, analogue, metabolite, prodrug orpharmaceutically acceptable salt thereof is formulated withcarboxymethyl cellulose to form a combined medicament.
 12. Apharmaceutical composition for use in the treatment of blood celldepletion, the composition comprising N⁶ isopentenyl adenosine or aderivative, analogue, metabolite, prodrug or pharmaceutically acceptablesalt thereof, along with a pharmaceutically acceptable diluent and/orcarrier.
 13. The pharmaceutical composition as claimed in claim 12wherein the composition is formulated with carboxymethyl cellulose. 14.The pharmaceutical composition as claimed in claim 12 further comprisingoltipraz or a derivative, analogue, metabolite, prodrug orpharmaceutically acceptable salt thereof.
 15. A combined pharmaceuticalcomposition for the prevention and/or treatment and/or amelioration of ablood cell deficiency disorder, such as neutropenia, thrombocytopenia oranaemia, the composition comprising a cytokinin compound and oltipraz ora derivative or functionally equivalent analogue thereof along with atleast one pharmaceutically acceptable diluent.
 16. The composition asclaimed in claim 15 wherein the cytokinin compound is N⁶ isopentenyladenosine or an analogue or a pharmaceutically acceptable salt thereof.17. The composition as claimed in claim 15 wherein the cytokinincompound is N⁶ benzyl adenosine or an analogue or pharmaceuticallyacceptable salt thereof.
 18. The composition as claimed in claim 15wherein the analogue or derivative of oltipraz is selected from thegroup consisting of 3H-1,2-dithiole-3-thione, sulforaphane, narigin,5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione, ADT, ADO,1,2-dithiole-3-thione, 1,2-dithiolane, 1,3-dithiole-2-thione andmalotilate.
 19. A method of stimulating cellular DNA repair mechanismsin a subject in advance of or following exposure of the subject toradiation, the method comprising the steps of: providing atherapeutically or prophylactically effective amount of oltipraz or aderivative, analogue, metabolite or salt thereof, and administering thecomposition to a subject in need thereof.
 20. The method as claimed inclaim 19 wherein the radiation exposure results from the subjectundergoing travel in space.
 21. The method as claimed in claim 19wherein the analogue or derivative of oltipraz is selected from thegroup consisting of 3H-1,2-dithiole-3-thione, sulforaphane, narigin,5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione, ADT, ADO,1,2-dithiole-3-thione, 1,2-dithiolane, 1,3-dithiole-2-thione andmalotilate.
 22. The method as claimed in claim 19 wherein the oltiprazmetabolite is metabolite
 3. 23. A pharmaceutical composition formediating DNA repair following radiation exposure comprising oltipraz ora derivative or analogue thereof along with a pharmaceuticallyacceptable carrier.
 24. The pharmaceutical composition as claimed inclaim 23 wherein the analogue or derivative of oltipraz is selected fromthe group consisting of 3H-1,2-dithiole-3-thione, sulforaphane, narigin,5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione, ADT, ADO,1,2-dithiole-3-thione, 1,2-dithiolane, 1,3-dithiole-2-thione andmalotilate.
 25. A method of treating and/or preventing radiation-inducedimmunosuppression, the method comprising the step of administering atherapeutically or prophylactically effective amount of N⁶ isopentenyladenosine to a subject in need thereof.